Journal: Gut microbes

Article Title: Gut microbiota metabolite indole-3-acetic acid maintains intestinal epithelial homeostasis through mucin sulfation.

doi: 10.1080/19490976.2024.2377576

Figure Lengend Snippet: Figure 4. IAA supplementation enhanced the mucosal barrier function by promoting mucin sulfation. (a) Representative micrographs of MUC2 immunohistochemistry (left panel) and quantification of MUC2 positive cells/crypt (right panel) in colon sections from different groups of mice. Scale bars: 30 µm. (b) Representative micrographs (left panel) and quantification (right panel) of AB-PAS staining (mucus layer was stained blue or purple) in distal colon sections from different groups of mice. Scale bars: 300 µm. (c) Representative micrographs (left panel) and quantification (right panel) of HID-AB staining (sulfomucin was stained brown) in distal colon sections from different groups of mice. Scale bars: 300 µm. (d) Immunofluorescence staining of DAPI (blue) and MALII lectin (green) in the distal colons (left panel) and quantification of the mean fluorescent intensity (MFI) (right panel). Scale bars: 50 µm. (e) The relative mRNA expression levels of Papss2, Slc35b3, and GlcNAc6st2 in the colon of mice by quantitative RT-PCR. (f) The protein expression of Papss2 in the colon of different groups of mice by western blot and quantification of the value of Papss2/β-actin. (g) Representative micrographs of immunohistochemical detection of Papss2 in mice colonic tissues (left panel) and quantification of the abundance of Papss2 (right panel). Scale bars: 30 µm. ns, not significant, *p < .05, **p < .01, ***p < .001.

Article Snippet: The membranes were blocked for 1 h with 10% nonfat milk, and then primary antibodies against Papss2 (1:500, sc -100,801, Santa Cruz), and β-actin (1:1000, Cat#mAb3700, CST) were added for 12 h at 4°C, followed by HRP-linked anti-mouse IgG (1:5000, Cat#7076, CST) for 1 h. Using the ImageJ software, the intensities of protein bands were measured and the values were adjusted to β-actin.

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemical staining

Journal: Gut microbes

Article Title: Gut microbiota metabolite indole-3-acetic acid maintains intestinal epithelial homeostasis through mucin sulfation.

doi: 10.1080/19490976.2024.2377576

Figure Lengend Snippet: Figure 6. IAA activated AHR to regulated mucin sulfation. (a) The relative mRNA expression levels of Papss2, Slc35b3, and GlcNAc6st2 in LS174T cells by quantitative RT-PCR. (b) The relative mRNA expression levels of Papss2, Slc35b3, and GlcNAc6st2 in HT-29 cells by quantitative RT-PCR. (c) Molecular docking analysis of IAA binding on the ligand-binding domain of AHR protein. The binding energy is −5.246 kcal/mol. (d) Immunostaining images of HT-29 cells stained for DAPI (blue), AHR (red), and merge. Scale bars: 50 µm. *p < .05, **p < .01, ***p < .001, ****p < .0001.

Article Snippet: The membranes were blocked for 1 h with 10% nonfat milk, and then primary antibodies against Papss2 (1:500, sc -100,801, Santa Cruz), and β-actin (1:1000, Cat#mAb3700, CST) were added for 12 h at 4°C, followed by HRP-linked anti-mouse IgG (1:5000, Cat#7076, CST) for 1 h. Using the ImageJ software, the intensities of protein bands were measured and the values were adjusted to β-actin.

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Ligand Binding Assay, Immunostaining, Staining

Journal: Gut microbes

Article Title: Gut microbiota metabolite indole-3-acetic acid maintains intestinal epithelial homeostasis through mucin sulfation.

doi: 10.1080/19490976.2024.2377576

Figure Lengend Snippet: Figure 8. AHR-XRE interaction mediated the effect of IAA on Papss2. (a) The relative mRNA expression levels of Papss2, Slc35b3 in Ahr−/− mice by quantitative RT-PCR. (b) Protein expression of Papss2 in Ahr−/− mice by western blot (left panel) and quantification of

Article Snippet: The membranes were blocked for 1 h with 10% nonfat milk, and then primary antibodies against Papss2 (1:500, sc -100,801, Santa Cruz), and β-actin (1:1000, Cat#mAb3700, CST) were added for 12 h at 4°C, followed by HRP-linked anti-mouse IgG (1:5000, Cat#7076, CST) for 1 h. Using the ImageJ software, the intensities of protein bands were measured and the values were adjusted to β-actin.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Gut microbes

Article Title: Gut microbiota metabolite indole-3-acetic acid maintains intestinal epithelial homeostasis through mucin sulfation.

doi: 10.1080/19490976.2024.2377576

Figure Lengend Snippet: Figure 10. Expression of Papss2 and mucin sulfation were down-regulated in UC patients exposed to HFD. (a) The gene expression of Papss2 and Slc35b3 in colonic mucosa from UC patients (n = 97), CD patients (n = 8) and normal individuals (n = 11) (GSE59071). (b) Single-cell RNA sequencing analysis reveals cell types annotated in colon tissue biopsy specimens. (c) The relative expression of Papss2 and Slc35b3 in goblet cells of inflammatory and non-inflammatory colon biopsy tissue samples from patients with UC. (d) Representative micrographs (left panel) and quantifications (right panel) of HID-AB staining (sulfomucin stains brown) in colon sections from UC patients with or without HFD diet. Scale bars: 50 µm. (e) Representative micrographs of immunohistochemical detection of Papss2 in colon sections from UC patients with or without HFD diet (left panel) and quantifications of the abundance of Papss2 (right panel). Scale bars: 50 µm. *p < .05, ***p < .001, ****p < .0001.

Article Snippet: The membranes were blocked for 1 h with 10% nonfat milk, and then primary antibodies against Papss2 (1:500, sc -100,801, Santa Cruz), and β-actin (1:1000, Cat#mAb3700, CST) were added for 12 h at 4°C, followed by HRP-linked anti-mouse IgG (1:5000, Cat#7076, CST) for 1 h. Using the ImageJ software, the intensities of protein bands were measured and the values were adjusted to β-actin.

Techniques: Expressing, Gene Expression, RNA Sequencing, Staining, Immunohistochemical staining